Cancer is an aberrant net accumulation of atypical cells, which can result from an excess of proliferation, an insufficiency of apoptosis, or a combination of the two. Apoptosis is a genetically programmed, non-inflammatory, energy-dependent form of cell death in tissue, including adult tissue (Steller H. Science 267:1445-1449, 1995), and is associated with nuclear DNA-fragmentation, release of nuclear matrix proteins (NuMA), and loss of cell substrate contact.
Apoptosis can be initiated by ligands which bind to cell surface receptors including, but not limited to, Fas (CD95) (French et al. Journal of Cell Biology 133:355-364, 1996) and tumor necrosis factor receptor 1 (TNFR1). FasL binding to Fas and TNF binding to TNFR1 initiate intracellular signaling resulting in the activation of cysteine aspartyl proteases (caspases), which initiate the lethal proteolytic cascade of apoptosis execution (Muzio et al. Cell 85:817-827, 1996). Mutations in Fas or in TNFR1 can cause a failure of apoptosis.
Apoptosis also can be induced by intracellular proteins including, but not limited to, p53/p21 regulators (Levine A. Cell 88:323-331, 1997). p53/p21 act as transcription factors to activate expression of apoptosis-mediating genes, including, but not limited to, genes encoding proteins that generate free radicals that, it turn, damage the cell's mitochondria, whose contents leak out into the cytoplasm and activate apoptotic caspases (Polyak et al. Nature 389:300-305, 1997). Loss of functional p53/p21 correlates with aggressiveness in a variety of cancers (Fisher D. Cell 78:529-542, 1994).
Resistance to apoptosis induction has emerged as an important category of multiple drug resistance (MDR), one that likely explains a significant proportion of treatment failures. MDR, the simultaneous resistance to structurally and functionally unrelated chemotherapeutic agents, can be both inherent and acquired. That is, some cancers never respond to therapy, whereas other cancers, initially sensitive to therapy, develop drug resistance. As chemotherapeutic agents rely primarily on induction of apoptosis in cancer cells for their therapeutic effect, drug resistance, which diminishes the effectiveness of chemotherapeutic agents, leads directly or indirectly to reduced apoptosis and is generally associated with poor prognosis in a variety of cancers.
Cancer of the bladder is particularly difficult to treat successfully (Lamm et al. Journal of Urology 153:1444-1450, 1995). Live bacillus Calmette-Guerin (BCG) has been shown to have activity against bladder cancer cells in vivo (Morales A. Journal of Urology 132:457-459, 1984) and in vitro (Jackson et al. International Journal of Oncology 5:697-703, 1994; Pryor et al. Cancer Immunology and Immunotherapy 41:309-316, 1995; Pryor et al. British Journal of Cancer 71:801-807, 1995). However, live BCG can cause serious side effects including, but not limited to, fever, serum sickness-like syndromes, granulomatous infection, sepsis and even death (Lamm et al. Journal of Urology 147:596-600, 1992). Moreover, variability in the immunogenicity and in the stability of both live BCG and heat-killed BCG make its use difficult and unpredictable.
Although BCG inhibits proliferation of bladder cancer cells, it does not directly induce apoptosis (Sasaki et al. Urology International 59:142-148, 1997), but does stimulate lymphocyte activated killer (LAK) cells and lymphocyte production of bioactive molecules (Kudoh et al. British Journal of Urology 80S2:40, 1997). Human bladder T24 cancer cells will undergo apoptosis after contact with LAK cells (Shemtov et al. Journal of Urology 154:269-274, 1995) and, will undergo apoptosis and cytolysis in the presence of bioactive molecules.
Cytolysis is the complete or partial destruction of a cell and is mediated by the immune system. As used herein, the immune system includes macrophages, monocytes, lymphocytes and leukocytes. Macrophages and monocytes in the bladder wall accumulate around tumor islands in patients with bladder cancer (El-Demiry et al. British Journal of Urology 58:436-442, 1986; loachim-Velogiammi et al. Journal of Pathology 174:183-189, 1994) and, when stimulated, produce bioactive molecules. By produce is meant synthesize and secrete. These bioactive molecules include, but are not limited reactive oxygen species and cytokines.
Reactive oxygen species include, but are not limited, to nitric oxide, superoxide radicals and hydroxyl radicals. Reactive oxygen species induce cytolysis and apoptosis in susceptible target cells. Cytokines include, but are not limited to, interleukin-1 (IL-1), interleukin-6 (IL-6), interleukin-10 (IL-10), interleukin-12 (IL-12), and GM-CSF. IL-12 is reported to have anti-cancer activity toward some cancer cell lines (Stine et al. Annals NY Academy of Science 795:420-421; 1996; Angillo et al. Annals NY Academy of Sciences 795:158-165, 1996; Chen et al. Journal of Immunology 159:351-359, 1997), whereas GM-CSF is reported to have pro-cancer activity toward some cancer cell lines (Hawkyard et al. Journal of Urology 150:514-518, 1993).
Preparations of bacterial origin, including, but not limited to, preparations from Mycobacterium species, have been used to treat cancers (U.S. Pat. No. 4,503,048). REGRESSIN.RTM., a non-viable mycobacterial cell wall extract (MCWE) formulated as a mineral oil emulsion (Bioniche, Inc. London, Ontario, Canada), has been shown to reduce cancer burden in bladder cancers (Kadhim et al. Journal of Urology 149:A255, 1996; Morales et al. Journal of Urology 157:A214, 1997). MCWE is composed primarily of peptidoglycan and glycolipid (Chin et al. Journal of Urology 156:1189-1193, 1996) and contain N-acetylmuramyl-L-alanyl-D-isoglutamine (muramyl dipeptide) and mycolic acid derivatives. Both muramyl dipeptide and mycolic acid derivatives stimulate the immune system by activation of macrophage and monocyte mediated reactions (Mallick et al. Comparative Immunology and Microbiology of Infectious Diseases 8:55-63, 1985; Teware et al. Veterinary Parasitology 62:223-230, 1996). However, the therapeutic benefits obtained using such cell wall extracts to treat cancer cells are variable and inconsistent, and appear to depend on the method by which the preparation is prepared and delivered, and on the stability of the preparation.
Most prior art anti-bladder cancer agents have proven to be less than adequate for clinical applications. Many of these agents are inefficient (Bischoff et al. Science 274:373-376, 1996) or toxic, have significant side effects (Lamm et al. Journal of Urology 153:14444-1450, 1995), result in development of drug resistance or immunosensitization, and are debilitating for the recipient. Moreover, many of these agents depend on Fas, TNFR1 or p53/p21 for their effectiveness.
Therefore, there is a need for a novel therapeutic agent that inhibits proliferation of and induces apoptosis in bladder cancer cells, and that stimulates responsive cells of the immune system to produce bioactive molecules. This therapeutic agent should be useful as an anti-bladder cancer agent and as an adjunct to other anti-bladder cancer agents. By adjunct is meant useful with other anti-bladder cancer agents to increase treatment effectiveness. Moreover, such a therapeutic agent should be simple and relatively inexpensive to prepare, its activity should be reproducible among preparations, its activity should remain stable over time, and its effects on bladder cancer cells should be achievable with dose regimens that are associated with minimal toxicity.